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Biological second and third harmonic generation microscopy.

Peter Friedl1, Katarina Wolf, Ulrich H von Andrian

  • 1University of Würzburg, Würzburg, Germany.

Current Protocols in Cell Biology
|January 30, 2008
PubMed
Summary
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Higher harmonic generation microscopy (HHGM) offers label-free, subcellular resolution imaging of biological tissues. This advanced multiphoton microscopy technique enables real-time, 4D reconstructions of cellular structures and dynamics.

Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Multiphoton microscopy is a standard for noninvasive imaging of thick specimens.
  • Higher harmonic generation microscopy (HHGM) utilizes nonlinear multiphoton excitation for contrast.

Purpose of the Study:

  • To present the principles of second and third harmonic generation (SHG, THG).
  • To describe the basic setup of a HHGM system.
  • To summarize current applications of HHGM in cell biology.

Main Methods:

  • HHGM relies on nonlinear multiphoton excitation.
  • HHGM generates second and third harmonic signals from ordered molecules without exogenous labels.
  • The technique allows for 3D and 4D reconstructions.

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Main Results:

  • HHGM provides detailed real-time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential.
  • It visualizes cell depolarization.
  • Applications in cell biology are summarized.

Conclusions:

  • HHGM is a powerful tool for label-free structural and molecular imaging of native samples.
  • Multimodal multiphoton microscopy combining HHGM and two-photon excited fluorescence will be key for real-time imaging of cell dynamics in live tissues.